T cell-mediated immune responses require contact-dependent information exchange between T cells and antigen (Ag)-presenting cells (APC). Naive T cells are primed by mature dendritic cells (DC) encountered in secondary lymphoid organs, such as peripheral lymph nodes (PLN). Naive T cells continuously recirculate between the blood and PLN, whereas DC collect Ag in peripheral tissues and then access to PLN via afferent lymphatics. Ag presentation by mature DC to naive CDS T cells in the PLN cortex induces T cell activation and proliferation. After a few days, the dividing cells differentiate into cytotoxic effector cells (CTL), which can kill APC. CTL activity is thought to be controlled by several mechanisms, including the action of regulatory T cells (Treg) and the propensity of CTL to undergo apoptosis upon withdrawal of survival signals. Thus, after the height of a CTL response, the pool of Ag-specific T cells contracts, leaving behind a small population of long-lived memory cells, which respond more vigorously than naive T cells when the Ag returns. Memory cells are heterogeneous with regard to their function and tissue distribution. The most immunoprotective subsets are the central memory cells (Tcm), which recirculate through PLN similar to naive T cells. It is widely held that these career decisions taken by T cells are regulated by the spatio-temporal arrangement of interacting communication molecules on the surface of T cells and APC. However, the physical nature and the kinetics of T cell-APC interactions in PLN are still largely unexplored. In preliminary work for this project, we have developed a new multiphoton intravital microscopy (MP-IVM) model to study APC and TCR transgenic CDS T cells in intact popliteal LN of anesthetized mice. This imaging approach produces 3D time-lapse movies of interacting T cells and APC at subcellular resolution and will be used to address the following three specific aims: 1.) To analyze the spatial, temporal and behavioral relationship between naive CDS T cells and DC in PLN;2.) To examine how Ag-experienced CTL and Tcm respond to re-encounter of Ag in PLN;and 3.) To explore the effect of Ag-specific Treg on CTL differentiation and function. The proposed experiments will generate a comprehensive, mechanism-oriented survey of the behavioral similarities and differences between distinct T cell subsets during primary and secondary immune responses. This information may lead to improved strategies for clinical immunomodulation, e.g. for vaccinations, tumor therapy and treatment of infectious, inflammatory and autoimmune diseases.